A survey of hemoglobin quaternary structures

We perform an analysis of the quaternary structure and dimer/dimer interface in the crystal structures of 165 human hemoglobin tetramers; 112 are in the T, 17 the R, 14 the Y (or R2) state; 11 are high-affinity T state mutants, and 11 may either be intermediates between the states, or off the allosteric transition pathway. The tertiary structure is fixed within each state, in spite of the different ligands, mutations, and chemical modifications present in individual entries. The geometry of the tetramer assembly is essentially the same in all the R or the Y state entries; it is slightly different in high salt and low salt crystals of T state hemoglobins. The dimer/dimer interface differs in terms of size, chemical composition and polar interactions, between the states. It is loosely packed, like crystal packing contacts or the subunit interface of weakly associated homodimers, and unlike most oligomeric proteins, which have close-packed interfaces. The loose packing is most obvious in the liganded forms, where the tetramer is known to dissociate at low concentration. We identify cavities that contribute to the loose packing of the α1β2 and α2β1 contacts. Two pairs of cavities occur recurrently in both the T and the R state tetramers. They may contribute to the allosteric mechanism by facilitating the subunit movements and the tertiary structure changes that accompany the transition from T to R to Y.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Energy minimization in low-frequency normal modes to efficiently allow for global flexibility during systematic protein-protein docking

Protein-protein association can frequently involve significant backbone conformational changes of the protein partners. A computationally rapid method has been developed that allows to approximately account for global conformational changes during systematic protein-protein docking starting from many thousands of start configurations. The approach employs precalculated collective degrees of freedom as additional variables during protein-protein docking minimization. The global collective degrees of freedom are obtained from normal mode analysis using a Gaussian network model for the protein. Systematic docking searches were performed on 10 test systems that differed in the degree of conformational change associated with complex formation and in the degree of overlap between observed conformational changes and precalculated flexible degrees of freedom. The results indicate that in case of docking searches that minimize the influence of local side chain conformational changes inclusion of global flexibility can significantly improve the agreement of the near-native docking solutions with the corresponding experimental structures. For docking of unbound protein partners in several cases an improved ranking of near native docking solutions was observed. This was achieved at a very modest ( approximately 2-fold) increase of computational demands compared to rigid docking. For several test cases the number of docking solutions close to experiment was also significantly enhanced upon inclusion of soft collective degrees of freedom. This result indicates that inclusion of global flexibility can facilitate in silico protein-protein association such that a greater number of different start configurations results in favorable complex formation.     

Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes

Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.     

Distinct affinity and effector residues in the binding site for a regulatory ligand

A hypothesis concerning two distinct classes of amino acid residues in some regulatory binding sites is proposed. The affinity residues are those that are unable to transduce the ligand information signal but are responsible for overcoming the barrier for the attachment of a ligand to its binding site while the effector residues transfer the binding signal to the other functional part of the protein, which then undergoes a non-equilibrium energetic cycle induced by interaction with the ligand.As an example, the purine nucleotide inhibition of H⁺ transport through the uncoupling protein of brown adipose tissue mitochondria is discussed; there is a concentration range in which the nucleotide is bound but does not inhibit H⁺ transport. This is interpreted in terms of inaccessibility of the effector residues inducing H⁺ transport inhibition below a certain threshold concentration.     

预测蛋白质—蛋白质复合物结构的软对接算法

提出了一种有效的软对接算法 ,用于在已知受体和配体三维结构的条件下预测蛋白质 蛋白质复合物的结构。该算法的分子模型基于Janin提出的简化蛋白质模型 ,并在此基础上有所改进。对蛋白质分子表面的柔性氨基酸残基Arg、Lys、Asp、Glu和Met进行了特殊处理 ,通过软化分子表面的方式考虑了它们的侧链柔性。采用双重过滤技术来排除不合理的对接结构 ,此过滤技术是以复合物界面几何互补性和残基成对偏好性为标准提出的。对所得到的构象进行能量优化 ,之后用打分函数对这些结构进行排序 ,挑选出与复合物天然结构接近的构象。该打分函数包括静电、疏水和范德华相互作用能。用此算法对 2 6个复合物进行了结构预测 ,均找到了近天然结构 ,其中有 2 0个复合物的近天然结构排在了前 10位。改进的分子模型可以在一定程度上描述蛋白质表面残基侧链的柔性 ;双重过滤技术使更多的近天然结构保留下来 ,从而提高了算法成功预测的可能性 ;打分函数可以较合理地评价对接结构。总之 ,此种软对接算法能够对蛋白质分子识别的研究提供有益的帮助。     

Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding.